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Abstract

Background

The chemokine receptor 1 CXCR-1 (or IL8R-alpha) is a specific receptor for the interleukin
8 (IL-8), which is chemoattractant for neutrophils and has an important role in the
inflammatory response. The polymorphism rs2234671 at position Ex2+860G > C of the
CXCR1 gene causes a conservative amino acid substitution (S276T). This single nucleotide
polymorphism (SNP) seemed to be functional as it was associated with decreased lung
cancer risk. Previous studies of our group found association of haplotypes in the
IL8 and in the CXCR2 genes with the multifactorial disease chronic periodontitis. In this study we investigated
the polymorphism rs2234671 in 395 Brazilian subjects with and without chronic periodontitis.

Findings

Similar distribution of the allelic and genotypic frequencies were observed between
the groups (p > 0.05).

Conclusions

The polymorphism rs2234671 in the CXCR1 gene was not associated with the susceptibility to chronic periodontitis in the studied
Brazilian population.

Keywords:

CXCR1; chemokine; cytokine; genetic polymorphism; periodontal disease

Findings

The human chemokine receptor CXCR-1 (or IL8R-alpha) is a specific receptor for the
chemokine interleukin 8 (IL-8) [1]. Initially identified as a chemoattractant for neutrophils, IL-8 has been demonstrated
to have pro-inflammatory effects including stimulation of neutrophil degranulation
[2]. Cellular activities of IL-8 are mediated by CXCR-1 and CXCR-2 (IL8R-beta), which
maintain 78% of amino acid similarity and are encoded by two single-copy genes that
are located on chromosome 2q34-35 [3]. However, CXCR-1 is more specific for IL-8 in comparison with CXCR-2 [4].

The involvement of IL-8, CXCR-1 and CXCR-2 has been extensively investigated in different
diseases such as pyelonephritis [5,6], hepatitis B [7], rapid disease progression of HIV-1+ [8], lung diseases, such as chronic obstructive pulmonary disease and asthma [9], bronchiectasis [10], systemic sclerosis [11] and lung cancer [12]. Some of these studies have reported positive associations between the diseases and
single nucleotide polymorphisms (SNPs) in the CXCR1 gene [8,9]. Indeed, a significant association was demonstrated between the 860G > C (S276T)
SNP in the CXCR1 gene with decreased lung cancer risk [12]. Haplotypes formed by SNPs in the CXCR1 and CXCR2 genes where also previously identified [7].

The 860G > C (S276T) SNP in CXCR1 gene was identified by comparison of multiple sequences deposited in the GenBank/EMBL
data banks [11,13]. These authors named this polymorphism differently: +2607 (position 6334 of sequence
accession number [GenBank: L19592.1]) in exon 2, and +827 (starting from the initiation of the ATG codon in exon 2 of
[GenBank: L19592.1]), respectively. The variant position 860G > C is based on the [NCBI: NM000634.2]
exon 2 initiations, however it is important to be clear that all these different positions
at the CXCR1 gene refer to the same polymorphism (G > C), which results in a conservative amino
acid substitution from serine to threonine at the 276 amino acid residue of the CXCR-1
(or IL8R-alpha) protein. Here, we preferred to use the reference sequence number [refSNP
ID: rs2234671] in NCBI's Entrez system (http://www.ncbi.nlm.nih.gov/SNPwebcite).

Predominantly in the last decade, many candidate-gene investigations have been conducted
in order to find genetic risk factors associated with chronic periodontitis (CP).
Epidemiological studies indicate that CP is an important cause of teeth loss in adults,
since 5% to 15% of any population suffers from this disease [14]. CP is a multifactorial disease, initiated by bacterial infection which can progress
to the damage and destruction of the supporting tissues of the teeth [15]. The host response is influenced by both environmental (e.g. smoking, oral hygiene)
and genetic factors [16]. Some studies have demonstrated association between polymorphisms in genes of the
immune system and CP, such as Interleukin 2 (IL2) [17,18], IL4 [19-21], IL6 [22,23] and IL10 [24,25]. Recently, we have reported association between haplotypes in the IL8 and in the CXCR2 genes with CP [26,27]. Because those previous findings and the biological relationship of the CXCR-1 with
the IL-8 and the CXCR-2 [1,5] we have hypothesized whether a SNP in the CXCR1 gene would also influence the host susceptibility to CP.

In this regard, the aim of this study was to investigate the association of the rs2234671
SNP in the CXCR1 gene in a Brazilian population with chronic periodontitis.

Materials and methods

In total, 395 individuals in good general health and having similar socioeconomic
status were recruited from the patient pool of the School of Dentistry at Araraquara,
São Paulo State University - UNESP between November 2004 and May 2007. The study was
approved by the Committee for Ethical Affairs of the São Paulo State University (Protocol
number 57/04).

To verify statistical power of our sample, we used the G*POWER 3 software [28] with the following parameters: logistic regression; two tail, odds ratio = 1.56,
α error probability = 0.05. Detailed clinical criteria for include patients in this
study are described in Viana et al. [27]. Briefly, the following clinical signs and parameters were assessed at six sites
around each tooth: probing pocket depth (PPD) (measured as distance from the free
gingival margin to the base of the pocket), clinical attachment loss (CAL) (as the
distance from the cement-enamel junction to the base of the periodontal pocket) and
bleeding on probing (BOP) (registered as present or absent). These measurements were
performed in millimeters using a periodontal probe with Williams markings. All fully
erupted teeth, except third molars and retained roots, were examined. The diagnosis
of subjects was established on the basis of clinical criteria proposed by the 1999
International World Workshop for a Classification of Periodontal Diseases and Conditions
[29]. The subjects were categorized into two groups: controls (n = 191), subjects exhibiting
no sites with CAL and PPD ≥ 3 mm and no BOP; and CP (n = 200), subjects exhibiting
one or more sites with CAL and PPD ≥ 3 mm and BOP.

Information on smoking status was obtained using a self-reported questionnaire, and
subjects were classified as a "smoker" or "nonsmoker" according to Kornman et al.[30].

The chi-squared test was used to determine whether the groups were composed of patients
with the same proportion of males and females and whether they had similar smoking
habits. Onset age distribution between the two groups was evaluated by Students't-test.
Those statistical analyses, as well as deviations from Hardy-Weinberg equilibrium,
were performed using BioEstat software version 5.0 (UFPA, MCT, CNPq, Belém, PA, Brazil).

In addition, a forward stepwise multiple logistic regression analysis was used for
estimating the relationships between the SNP rs2234671 in the CXCR1gene and periodontal disease susceptibility and the other covariates. This multivariate
logistic regression modeling was executed using the R statistical package (R Development
Core Team, Vienna, Austria). Differences were considered to be significant when p
< 0.05.

Results

The power calculations showed that the sample size of 395 individuals demonstrated
a power of 93%. Therefore, the number of subjects enrolled in this study is large
enough to detect association with an acceptable level of confidence. The population
investigated here was composed mainly of female subjects (59.7%) and nonsmokers (84.8%)
and the mean age of the individuals was 39.49 years (Table 1). The minor allele frequency (MAF) was 0.087, and the genotype distribution of the
rs2234671 SNP in the CXCR1 gene was consistent with the assumption of the Hardy-Weinberg equilibrium for the
control (p = 0.1719) and CP groups (p = 0.2777).

Significant associations between age and periodontitis were observed by univariate
analysis, mainly considering the ORs for age groups 30-39 to the 50-59. We consider
that besides the 60-69 and > 70 age groups showed significant OR values they were
uncommon and demonstrated a wide range of CI. Significant association was also found
between smoking status and periodontitis (OR = 3.8). Therefore, age and smoking status
were considered confounding factors. Even though gender was not found to be associated
with periodontitis, we included it in the confounding factors in the multivariate
analysis, to adjust for any small confounding effects. To more accurately evaluate
the strength of any association and to eliminate the distortion caused by confounding
effects, multivariate analysis was performed. The multiple logistic regression analysis
demonstrated that neither genotypes nor alleles were associated with periodontitis,
even after adjusting for covariates, including age, gender and smoking status (Table
2). Therefore, this polymorphism could not be considered a genetic risk for CP in the
studied Brazilian population.

Discussion

To our knowledge, this is the first time that the rs2234671 SNP in the CXCR1 gene was investigated regarding the CP. Single nucleotide polymorphisms are mainly
useful in studies of human population genetics and candidate-gene studies for disease
association [31]. This makes possible the development of the susceptibility profile concept for specific
diseases, like the risk of Alzheimer's, which has been considered to be substantially
influenced by a total of ten genetic polymorphisms of inflammation-related molecules
[32]. If the high-susceptibility profile for CP be determined, genetically susceptible
subjects would be identified earlier and therapeutic intervention strategies could
be envisaged aiming prevention of disease establishment. However, many studies with
different populations would be needed to reach a high-risk profile for periodontal
disease[16].

Another important point to be considered in studying genetic variations is about the
functionality of a polymorphism. Considering that the rs2234671 SNP causes a conservative
amino acid substitution (S276T) in the third extracellular loop of the CXCR-1 protein,
one can suppose whether this event would influence the ligand/binding interaction
of the chemokine receptor. This speculation was raised by Liu and colleagues [33], which found four SNPs (including the rs2234671) in human populations, that were
not found in nonhuman primate species when the gene sequences were compared. The authors
concluded that there is an accelerated CXCR-1 protein evolution in the human lineage.
However, more studies are necessary in order to clarify whether this SNP is functional,
and whether it would be under selective pressure aiming to improve innate immunity.

Relevant factors known to influence the pathogenesis of periodontitis were assessed
by multivariate analysis. Regarding age, there is evidence that both the prevalence
and severity of periodontitis increase with increasing age [34]. This can be explained by the cumulative effect of prolonged exposure to other risk
factors [35]. There is a multitude of studies (more than 325) that have shown a relationship between
smoking and periodontitis [36]. In the present study, smoking was found as a risk factor for periodontitis (OR =
3.8, 95% CI: 1.97-7.33). Interestingly, Tonetti et al.[37] reported that cigarette smoking is associated with two- to threefold increases in
the odds of developing periodontitis. Also similar with our results, Tomar & Asma
[38] found that current smokers were about four times as likely to have periodontitis
than persons who never smoked, after adjusting for covariates such as age, gender
and education. Some studies have shown that smokers had significantly worse clinical
symptoms of periodontitis than non-smokers [39,40].

We did not take into account ancestry in our study because we only obtained the skin
color information. Although Brazilian population is admixtured, the skin color is
not related with ancestry according to Pena et al.[41], Parra et al.[42] and Pimenta et al.[43].

In regards to our hypothesis whether the rs2234671 SNP in the CXCR1 gene would be associated with susceptibility to chronic periodontitis, the obtained
results showing a lack of association permit us to conclude that this SNP was not
useful as a genetic risk factor for CP in the studied Brazilian population.

Abbreviations

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

RMSC conceived and coordinated the study, performed statistical analysis and wrote
the manuscript. KMCC, RR, PMS: carried out the molecular reactions and helped analyze
the results. GA: helped with the statistical analysis and helped wrote the manuscript.
ACV, YJK: examined and selected the patients and extracted the DNA. SRPO, JAC: participated
in the study design, defined the clinical criteria of the studied groups and helped
wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements

This study was supported by CAPES and FAPESP grants (03/10424-0, 06/04492-1, 05/03231-7,
2005/04553-8).

Trevilatto PC, Scarel-Caminaga RM, de Brito RB, de Souza AP, Line SR: Polymorphism at position -174 of IL-6 gene is associated with susceptibility to chronic
periodontitis in a Caucasian Brazilian population.